Article | Published:

Genome-wide analysis reveals characteristics of off-target sites bound by the Cas9 endonuclease

Nature Biotechnology volume 32, pages 677683 (2014) | Download Citation

Abstract

RNA-guided genome editing with the CRISPR-Cas9 system has great potential for basic and clinical research, but the determinants of targeting specificity and the extent of off-target cleavage remain insufficiently understood. Using chromatin immunoprecipitation and high-throughput sequencing (ChIP-seq), we mapped genome-wide binding sites of catalytically inactive Cas9 (dCas9) in HEK293T cells, in combination with 12 different single guide RNAs (sgRNAs). The number of off-target sites bound by dCas9 varied from 10 to >1,000 depending on the sgRNA. Analysis of off-target binding sites showed the importance of the PAM-proximal region of the sgRNA guiding sequence and that dCas9 binding sites are enriched in open chromatin regions. When targeted with catalytically active Cas9, some off-target binding sites had indels above background levels in a region around the ChIP-seq peak, but generally at lower rates than the on-target sites. Our results elucidate major determinants of Cas9 targeting, and we show that ChIP-seq allows unbiased detection of Cas9 binding sites genome-wide.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Accessions

Primary accessions

Gene Expression Omnibus

Referenced accessions

Gene Expression Omnibus

References

  1. 1.

    et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013).

  2. 2.

    et al. RNA-programmed genome editing in human cells. eLife 2, e00471 (2013).

  3. 3.

    , , , & RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat. Biotechnol. 31, 233–239 (2013).

  4. 4.

    et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).

  5. 5.

    et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).

  6. 6.

    & CRISPR/Cas, the immune system of bacteria and archaea. Science 327, 167–170 (2010).

  7. 7.

    et al. Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acids Res. 41, 4336–4343 (2013).

  8. 8.

    et al. Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat. Biotechnol. 31, 227–229 (2013).

  9. 9.

    et al. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153, 910–918 (2013).

  10. 10.

    , , , & Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library. Nat. Biotechnol. 32, 267–273 (2014).

  11. 11.

    , , & Genetic screens in human cells using the CRISPR-Cas9 system. Science 343, 80–84 (2014).

  12. 12.

    et al. Genome-scale CRISPR-Cas9 knockout screening in human cells. Science 343, 84–87 (2014).

  13. 13.

    et al. RNA-guided gene activation by CRISPR-Cas9-based transcription factors. Nat. Methods 10, 973–976 (2013).

  14. 14.

    et al. One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering. Cell 154, 1370–1379 (2013).

  15. 15.

    et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152, 1173–1183 (2013).

  16. 16.

    et al. CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat. Biotechnol. 31, 833–838 (2013).

  17. 17.

    et al. Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system. Cell 155, 1479–1491 (2013).

  18. 18.

    & 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, 132–136 (2013).

  19. 19.

    et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol. 31, 827–832 (2013).

  20. 20.

    et al. High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nat. Biotechnol. 31, 839–843 (2013).

  21. 21.

    et al. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat. Biotechnol. 31, 822–826 (2013).

  22. 22.

    et al. Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases. Genome Res. 24, 132–141 (2014).

  23. 23.

    The ENCODE Project Consortium. et al. An integrated encyclopedia of DNA elements in the human genome. Nature 489, 57–74 (2012).

  24. 24.

    , & Genome-wide chromatin maps derived from limited numbers of hematopoietic progenitors. Nat. Methods 7, 615–618 (2010).

  25. 25.

    et al. Measuring chromatin interaction dynamics on the second time scale at single-copy genes. Science 342, 369–372 (2013).

  26. 26.

    et al. PatMaN: rapid alignment of short sequences to large databases. Bioinformatics 24, 1530–1531 (2008).

  27. 27.

    et al. Model-based analysis of ChIP-seq (MACS). Genome Biol. 9, R137 (2008).

  28. 28.

    et al. Genome-wide mapping of DNase hypersensitive sites using massively parallel signature sequencing (MPSS). Genome Res. 16, 123–131 (2006).

  29. 29.

    et al. Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced Genome Editing Specificity. Cell 154, 1380–1389 (2013).

  30. 30.

    & Whole-genome chromatin profiling from limited numbers of cells using nano-ChIP-seq. Nat. Protoc. 6, 1656–1668 (2011).

  31. 31.

    , , & Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009).

  32. 32.

    et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).

  33. 33.

    & BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26, 841–842 (2010).

  34. 34.

    & Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012).

  35. 35.

    et al. Integrative genomics viewer. Nat. Biotechnol. 29, 24–26 (2011).

  36. 36.

    et al. Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities. Mol. Cell 38, 576–589 (2010).

Download references

Acknowledgements

The research was funded with a departmental startup fund from the University of Virginia and from an American Cancer Society institutional research grant. We would like to thank A. Quinlan, University of Virginia, for technical help with data analysis and D. Burke, University of Virginia, for his critical readings and comments.

Author information

Author notes

    • Cem Kuscu
    •  & Sevki Arslan

    These authors contributed equally to this work.

Affiliations

  1. School of Medicine, Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, Virginia, USA.

    • Cem Kuscu
    • , Sevki Arslan
    • , Ritambhara Singh
    • , Jeremy Thorpe
    •  & Mazhar Adli
  2. Department of Biology, Pamukkale University, Denizli, Turkey.

    • Sevki Arslan
  3. Department of Computer Science, University of Virginia, Charlottesville, Virginia, USA.

    • Ritambhara Singh

Authors

  1. Search for Cem Kuscu in:

  2. Search for Sevki Arslan in:

  3. Search for Ritambhara Singh in:

  4. Search for Jeremy Thorpe in:

  5. Search for Mazhar Adli in:

Contributions

M.A. designed the study and wrote the manuscript. C.K. and S.A. performed the experiments. J.T. helped with experiments. R.S., C.K. and S.A. analyzed the data.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Mazhar Adli.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–7 and Supplementary Tables 1–7

Excel files

  1. 1.

    Supplementary Data

    dCas9 binding sites mediated by 12 sgRNAs (sgRNA guiding sequence matched bases at off-targets) sorted according to the MACs14 peak score

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nbt.2916

Further reading