Skip to main content
SpringerLink
Log in
Book cover

CRISPR Guide RNA Design pp 233–242Cite as

Profiling Genome-Wide Specificity of CRISPR-Cas9 Using Digenome-Seq

  • Protocol
  • First Online:

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2162))

Abstract

Digenome-seq is a highly sensitive method for analyzing the genome-wide specificity of CRISPR-Cas9 nuclease activity. In this procedure, genomic DNA is first subjected to digestion by CRISPR-Cas9 in vitro and then to whole genome sequencing, which results in unusual patterns of straight alignments at on-target and potential off-target sites. Analysis of these data with the Digenome-seq computer program allows for identification of the in vitro cleavage sites associated with the straight alignments. Here, we present a detailed Digenome-seq protocol for genome-wide profiling of in vitro CRISPR-Cas9 nuclease cleavage sites.

This is a preview of subscription content, log in via an institution.

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   109.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   139.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   249.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Bibikova M, Beumer K, Trautman JK, Carroll D (2003) Enhancing gene targeting with designed zinc finger nucleases. Science 300(5620):764. https://doi.org/10.1126/science.1079512

    Article  CAS  PubMed  Google Scholar 

  2. Kim HJ, Lee HJ, Kim H, Cho SW, Kim JS (2009) Targeted genome editing in human cells with zinc finger nucleases constructed via modular assembly. Genome Res 19(7):1279–1288. https://doi.org/10.1101/gr.089417.108

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Porteus MH, Baltimore D (2003) Chimeric nucleases stimulate gene targeting in human cells. Science 300(5620):763. https://doi.org/10.1126/science.1078395

    Article  PubMed  Google Scholar 

  4. Urnov FD, Miller JC, Lee YL, Beausejour CM, Rock JM, Augustus S, Jamieson AC, Porteus MH, Gregory PD, Holmes MC (2005) Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature 435(7042):646–651. https://doi.org/10.1038/nature03556

    Article  CAS  PubMed  Google Scholar 

  5. Kim Y, Kweon J, Kim A, Chon JK, Yoo JY, Kim HJ, Kim S, Lee C, Jeong E, Chung E, Kim D, Lee MS, Go EM, Song HJ, Kim H, Cho N, Bang D, Kim S, Kim JS (2013) A library of TAL effector nucleases spanning the human genome. Nat Biotechnol 31(3):251–258. https://doi.org/10.1038/nbt.2517

    Article  CAS  PubMed  Google Scholar 

  6. Kim YK, Wee G, Park J, Kim J, Baek D, Kim JS, Kim VN (2013) TALEN-based knockout library for human microRNAs. Nat Struct Mol Biol 20(12):1458–1464. https://doi.org/10.1038/nsmb.2701

    Article  CAS  PubMed  Google Scholar 

  7. Miller JC, Tan S, Qiao G, Barlow KA, Wang J, Xia DF, Meng X, Paschon DE, Leung E, Hinkley SJ, Dulay GP, Hua KL, Ankoudinova I, Cost GJ, Urnov FD, Zhang HS, Holmes MC, Zhang L, Gregory PD, Rebar EJ (2011) A TALE nuclease architecture for efficient genome editing. Nat Biotechnol 29(2):143–148. https://doi.org/10.1038/nbt.1755

    Article  CAS  PubMed  Google Scholar 

  8. Cho SW, Kim S, Kim JM, Kim JS (2013) Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat Biotechnol 31(3):230–232. https://doi.org/10.1038/nbt.2507

    Article  CAS  PubMed  Google Scholar 

  9. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339(6121):819–823. https://doi.org/10.1126/science.1231143

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Jinek M, East A, Cheng A, Lin S, Ma E, Doudna J (2013) RNA-programmed genome editing in human cells. eLife 2:e00471. https://doi.org/10.7554/eLife.00471

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM (2013) RNA-guided human genome engineering via Cas9. Science 339(6121):823–826. https://doi.org/10.1126/science.1232033

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Kim H, Kim JS (2014) A guide to genome engineering with programmable nucleases. Nat Rev Genet 15(5):321–334. https://doi.org/10.1038/nrg3686

    Article  CAS  PubMed  Google Scholar 

  13. Cho SW, Kim S, Kim Y, Kweon J, Kim HS, Bae S, Kim JS (2014) Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases. Genome Res 24(1):132–141. https://doi.org/10.1101/gr.162339.113

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Cradick TJ, Fine EJ, Antico CJ, Bao G (2013) CRISPR/Cas9 systems targeting beta-globin and CCR5 genes have substantial off-target activity. Nucleic Acids Res 41(20):9584–9592. https://doi.org/10.1093/nar/gkt714

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Fu Y, Foden JA, Khayter C, Maeder ML, Reyon D, Joung JK, Sander JD (2013) High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat Biotechnol 31(9):822–826. https://doi.org/10.1038/nbt.2623

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Hsu PD, Scott DA, Weinstein JA, Ran FA, Konermann S, Agarwala V, Li Y, Fine EJ, Wu X, Shalem O, Cradick TJ, Marraffini LA, Bao G, Zhang F (2013) DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol 31(9):827–832. https://doi.org/10.1038/nbt.2647

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Pattanayak V, Lin S, Guilinger JP, Ma E, Doudna JA, Liu DR (2013) High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nat Biotechnol 31(9):839–843. https://doi.org/10.1038/nbt.2673

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Kim D, Bae S, Park J, Kim E, Kim S, Yu HR, Hwang J, Kim JI, Kim JS (2015) Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells. Nat Methods 12(3):231–243. https://doi.org/10.1038/nmeth.3284

    Article  CAS  Google Scholar 

  19. Kim D, Kim S, Kim S, Park J, Kim JS (2016) Genome-wide target specificities of CRISPR-Cas9 nucleases revealed by multiplex digenome-seq. Genome Res 26(3):406–415. https://doi.org/10.1101/gr.199588.115

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. 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 

  21. Kim E, Koo T, Park SW, Kim D, Kim K, Cho HY, Song DW, Lee KJ, Jung MH, Kim S, Kim JH, Kim JH, Kim JS (2017) In vivo genome editing with a small Cas9 orthologue derived from Campylobacter jejuni. Nat Commun 8:14500. https://doi.org/10.1038/ncomms14500

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Endo M, Mikami M, Endo A, Kaya H, Itoh T, Nishimasu H, Nureki O, Toki S (2018) Genome editing in plants by engineered CRISPR-Cas9 recognizing NG PAM. Nat Plants. https://doi.org/10.1038/s41477-018-0321-8

  23. Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc 3(6):1101–1108

    Article  CAS  PubMed  Google Scholar 

  24. Kim D, Lim K, Kim ST, Yoon SH, Kim K, Ryu SM, Kim JS (2017) Genome-wide target specificities of CRISPR RNA-guided programmable deaminases. Nat Biotechnol 35(5):475–480. https://doi.org/10.1038/nbt.3852

    Article  CAS  PubMed  Google Scholar 

  25. Park J, Childs L, Kim D, Hwang GH, Kim S, Kim ST, Kim JS, Bae S (2017) Digenome-seq web tool for profiling CRISPR specificity. Nat Methods 14(6):548–549. https://doi.org/10.1038/nmeth.4262

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Center for Genome Engineering, Institute for Basic Science (IBS), Seoul, Republic of Korea

    Daesik Kim

  2. Genome Editing Research Center, KRIBB, Daejeon, Republic of Korea

    Daesik Kim

  3. Center for Genome Engineering, Institute for Basic Science (IBS), Seoul, South Korea

    Jin-Soo Kim

  4. Department of Chemistry, Seoul National University, Seoul, South Korea

    Jin-Soo Kim

Authors
  1. Daesik Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jin-Soo Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jin-Soo Kim .

Editor information

Editors and Affiliations

  1. Radcliffe Department of Medicine, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK

    Tudor A. Fulga

  2. Radcliffe Department of Medicine, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK

    David J. H. F. Knapp

  3. Radcliffe Department of Medicine, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK

    Quentin R. V. Ferry

Rights and permissions

Reprints and permissions

Copyright information

© 2021 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Kim, D., Kim, JS. (2021). Profiling Genome-Wide Specificity of CRISPR-Cas9 Using Digenome-Seq. In: Fulga, T.A., Knapp, D.J.H.F., Ferry, Q.R.V. (eds) CRISPR Guide RNA Design. Methods in Molecular Biology, vol 2162. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0687-2_13

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-0687-2_13

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0686-5

  • Online ISBN: 978-1-0716-0687-2

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation