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Dissecting Locus-Specific Chromatin Interactions by CRISPR CAPTURE

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DNA-Protein Interactions

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2599))

Abstract

The spatiotemporal control of tissue-specific gene expression is coordinated by cis-regulatory elements (CREs) and associated trans-acting factors. Despite major advances in genome-wide annotation of candidate CREs, the in situ regulatory composition of the vast majority of CREs remain unknown. To address this challenge, we developed the CRISPR affinity purification in situ of regulatory elements (CAPTURE) toolbox that employs an in vivo biotinylated nuclease-deficient Cas9 (dCas9) protein and programmable single-guide RNAs (sgRNAs) to identify CRE-associated macromolecular complexes and chromatin looping. In this chapter, we provide a detailed protocol for implementing the latest iteration of the CRISPR-based CAPTURE methods to interrogate the molecular composition of locus-specific chromatin complexes and configuration in a mammalian genome.

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References

  1. Lupiáñez DG, Kraft K, Heinrich V, Krawitz P, Brancati F, Klopocki E, Horn D, Kayserili H, Opitz JM, Laxova R, Santos-Simarro F, Gilbert-Dussardier B, Wittler L, Borschiwer M, Haas SA, Osterwalder M, Franke M, Timmermann B, Hecht J, Spielmann M, Visel A, Mundlos S (2015) Disruptions of topological chromatin domains cause pathogenic rewiring of gene-enhancer interactions. Cell 161(5):1012–1025. https://doi.org/10.1016/j.cell.2015.04.004

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Hnisz D, Weintraub AS, Day DS, Valton AL, Bak RO, Li CH, Goldmann J, Lajoie BR, Fan ZP, Sigova AA, Reddy J, Borges-Rivera D, Lee TI, Jaenisch R, Porteus MH, Dekker J, Young RA (2016) Activation of proto-oncogenes by disruption of chromosome neighborhoods. Science 351(6280):1454–1458. https://doi.org/10.1126/science.aad9024

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Li K, Zhang Y, Liu X, Liu Y, Gu Z, Cao H, Dickerson KE, Chen M, Chen W, Shao Z, Ni M, Xu J (2020) Noncoding variants connect enhancer dysregulation with nuclear receptor signaling in hematopoietic malignancies. Cancer Discov 10(5):724–745. https://doi.org/10.1158/2159-8290.CD-19-1128

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Ren B, Robert F, Wyrick JJ, Aparicio O, Jennings EG, Simon I, Zeitlinger J, Schreiber J, Hannett N, Kanin E, Volkert TL, Wilson CJ, Bell SP, Young RA (2000) Genome-wide location and function of DNA binding proteins. Science 290(5500):2306–2309. https://doi.org/10.1126/science.290.5500.2306

    Article  CAS  PubMed  Google Scholar 

  5. Griesenbeck J, Boeger H, Strattan JS, Kornberg RD (2003) Affinity purification of specific chromatin segments from chromosomal loci in yeast. Mol Cell Biol 23(24):9275–9282. https://doi.org/10.1128/MCB.23.24.9275-9282.2003

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Déjardin J, Kingston RE (2009) Purification of proteins associated with specific genomic Loci. Cell 136(1):175–186. https://doi.org/10.1016/j.cell.2008.11.045

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Fujita T, Fujii H (2011) Direct identification of insulator components by insertional chromatin immunoprecipitation. PLoS One 6(10):e26109. https://doi.org/10.1371/journal.pone.0026109

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Agelopoulos M, McKay DJ, Mann RS (2012) Developmental regulation of chromatin conformation by Hox proteins in Drosophila. Cell Rep 1(4):350–359. https://doi.org/10.1016/j.celrep.2012.03.003

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Fujita T, Fujii H (2013) Efficient isolation of specific genomic regions and identification of associated proteins by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using CRISPR. Biochem Biophys Res Commun 439(1):132–136. https://doi.org/10.1016/j.bbrc.2013.08.013

    Article  CAS  PubMed  Google Scholar 

  10. Waldrip ZJ, Byrum SD, Storey AJ, Gao J, Byrd AK, Mackintosh SG, Wahls WP, Taverna SD, Raney KD, Tackett AJ (2014) A CRISPR-based approach for proteomic analysis of a single genomic locus. Epigenetics 9(9):1207–1211. https://doi.org/10.4161/epi.29919

    Article  PubMed  PubMed Central  Google Scholar 

  11. Liu X, Zhang Y, Chen Y, Li M, Zhou F, Li K, Cao H, Ni M, Liu Y, Gu Z, Dickerson KE, Xie S, Hon GC, Xuan Z, Zhang MQ, Shao Z, Xu J (2017) In situ capture of chromatin interactions by biotinylated dCas9. Cell 170(5):1028–1043.e19. https://doi.org/10.1016/j.cell.2017.08.003

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Liu X, Zhang Y, Chen Y, Li M, Shao Z, Zhang MQ, Xu J (2018) CAPTURE: in situ analysis of chromatin composition of endogenous genomic loci by biotinylated dCas9. Curr Protoc Mol Biol 123(1):e64. https://doi.org/10.1002/cpmb.64

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Liu X, Chen Y, Zhang Y, Liu Y, Liu N, Botten GA, Cao H, Orkin SH, Zhang MQ, Xu J (2020) Multiplexed capture of spatial configuration and temporal dynamics of locus-specific 3D chromatin by biotinylated dCas9. Genome Biol 21(1):59. https://doi.org/10.1186/s13059-020-01973-w

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Burgess DJ (2017) Technique: CRISPR CAPTURE for multi-omic probing of genomic loci. Nat Rev Genet 18(11):641. https://doi.org/10.1038/nrg.2017.79

    Article  CAS  PubMed  Google Scholar 

  15. Alekseyenko AA, McElroy KA, Kang H, Zee BM, Kharchenko PV, Kuroda MI (2015) BioTAP-XL: cross-linking/tandem affinity purification to study DNA targets, RNA, and protein components of chromatin-associated complexes. Curr Protoc Mol Biol 109:21.30.1–21.30.32. https://doi.org/10.1002/0471142727.mb2130s109

    Article  PubMed  Google Scholar 

  16. Schatz PJ (1993) Use of peptide libraries to map the substrate specificity of a peptide-modifying enzyme: a 13 residue consensus peptide specifies biotinylation in Escherichia coli. Bio/technology (Nature Publishing Company) 11(10):1138–1143. https://doi.org/10.1038/nbt1093-1138

    Article  CAS  PubMed  Google Scholar 

  17. Buenrostro JD, Giresi PG, Zaba LC, Chang HY, Greenleaf WJ (2013) Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Nat Methods 10(12):1213–1218. https://doi.org/10.1038/nmeth.2688

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Concordet JP, Haeussler M (2018) CRISPOR: intuitive guide selection for CRISPR/Cas9 genome editing experiments and screens. Nucleic Acids Res 46(W1):W242–W245. https://doi.org/10.1093/nar/gky354

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Robinson JT, Thorvaldsdottir H, Winckler W, Guttman M, Lander ES, Getz G, Mesirov JP (2011) Integrative genomics viewer. Nat Biotechnol 29(1):24–26. https://doi.org/10.1038/nbt.1754

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Chen B, Gilbert LA, Cimini BA, Schnitzbauer J, Zhang W, Li GW, Park J, Blackburn EH, Weissman JS, Qi LS, Huang B (2013) Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system. Cell 155(7):1479–1491. https://doi.org/10.1016/j.cell.2013.12.001

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Marcel M (2011) Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet J 17(1):10–12. https://doi.org/10.14806/ej.17.1.200

    Article  Google Scholar 

  22. Langmead B, Salzberg SL (2012) Fast gapped-read alignment with Bowtie 2. Nat Methods 9(4):357–359. https://doi.org/10.1038/nmeth.1923

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Quinlan AR, Hall IM (2010) BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26(6):841–842. https://doi.org/10.1093/bioinformatics/btq033

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Zhang Y, Liu T, Meyer CA, Eeckhoute J, Johnson DS, Bernstein BE, Nusbaum C, Myers RM, Brown M, Li W, Liu XS (2008) Model-based analysis of ChIP-Seq (MACS). Genome Biol 9(9):R137. https://doi.org/10.1186/gb-2008-9-9-r137

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Tu S, Li M, Chen H, Tan F, Xu J, Waxman DJ, Zhang Y, Shao Z (2021) MAnorm2 for quantitatively comparing groups of ChIP-seq samples. Genome Res 31(1):131–145. https://doi.org/10.1101/gr.262675.120

    Article  PubMed  PubMed Central  Google Scholar 

  26. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R, 1000 Genome Project Data Processing Subgroup (2009) The sequence alignment/map format and SAM tools. Bioinformatics 25(16):2078–2079. https://doi.org/10.1093/bioinformatics/btp352

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Shariati SA, Dominguez A, Xie S, Wernig M, Qi LS, Skotheim JM (2019) Reversible disruption of specific transcription factor-DNA interactions using CRISPR/Cas9. Mol Cell 74(3):622–633.e4. https://doi.org/10.1016/j.molcel.2019.04.011

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Krijger P, Geeven G, Bianchi V, Hilvering C, de Laat W (2020) 4C-seq from beginning to end: a detailed protocol for sample preparation and data analysis. Methods 170:17–32. https://doi.org/10.1016/j.ymeth.2019.07.014

    Article  CAS  PubMed  Google Scholar 

  29. Cha HJ, Uyan Ö, Kai Y, Liu T, Zhu Q, Tothova Z, Botten GA, Xu J, Yuan GC, Dekker J, Orkin SH (2021) Inner nuclear protein Matrin-3 coordinates cell differentiation by stabilizing chromatin architecture. Nat Commun 12(1):6241. https://doi.org/10.1038/s41467-021-26574-4

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Li X, Burnight ER, Cooney AL, Malani N, Brady T, Sander JD, Staber J, Wheelan SJ, Joung JK, McCray PB Jr, Bushman FD, Sinn PL, Craig NL (2013) piggyBac transposase tools for genome engineering. Proc Natl Acad Sci U S A 110(25):E2279–E2287. https://doi.org/10.1073/pnas.1305987110

    Article  PubMed  PubMed Central  Google Scholar 

  31. Herbst F, Ball CR, Tuorto F, Nowrouzi A, Wang W, Zavidij O, Dieter SM, Fessler S, van der Hoeven F, Kloz U, Lyko F, Schmidt M, von Kalle C, Glimm H (2012) Extensive methylation of promoter sequences silences lentiviral transgene expression during stem cell differentiation in vivo. Mol Ther 20(5):1014–1021. https://doi.org/10.1038/mt.2012.46

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Li D, Hsu S, Purushotham D, Sears RL, Wang T (2019) WashU epigenome browser update 2019. Nucleic Acids Res 47(W1):W158–W165. https://doi.org/10.1093/nar/gkz348

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgments

We thank Drs. Xin Liu, Yuannyu Zhang, Yong Chen, Mushan Li, Zhen Shao, and Michael Q. Zhang for their help with the assay development and/or data analysis pipelines, Yoon Jung Kim at the Children’s Research Institute (CRI) Sequencing Core Facility for assistance with next-generation sequencing, and other Xu laboratory members for discussion and technical support. G.A.B. was supported by the American Heart Association (AHA) Predoctoral Fellowship (827234). J.X. is a Scholar of The Leukemia & Lymphoma Society (LLS) and an American Society of Hematology (ASH) Scholar. This work was supported by the NIH grants R01DK111430, R01CA230631, R01CA259581, and R21AI158240 (to J.X.), by the CPRIT grants (RP180504 and RP190417 to J.X.), and by the Welch Foundation grants I-1942 (to J.X.).

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Authors and Affiliations

  1. Children’s Medical Center Research Institute, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Giovanni A. Botten, Michael Lee Jr & Jian Xu

  2. Department of Pediatrics, Harold C. Simmons Comprehensive Cancer Center, and Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Giovanni A. Botten, Michael Lee Jr & Jian Xu

Authors
  1. Giovanni A. Botten
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  2. Michael Lee Jr
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  3. Jian Xu
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Corresponding author

Correspondence to Jian Xu .

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Editors and Affiliations

  1. Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, USA

    Marcos Simoes-Costa

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Botten, G.A., Lee, M., Xu, J. (2023). Dissecting Locus-Specific Chromatin Interactions by CRISPR CAPTURE. In: Simoes-Costa, M. (eds) DNA-Protein Interactions. Methods in Molecular Biology, vol 2599. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2847-8_7

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  • DOI: https://doi.org/10.1007/978-1-0716-2847-8_7

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2846-1

  • Online ISBN: 978-1-0716-2847-8

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