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CIRCLE-seq: a highly sensitive in vitro screen for genome-wide CRISPR–Cas9 nuclease off-targets

Nature Methods volume 14, pages 607614 (2017) | Download Citation

  • A Corrigendum to this article was published on 27 April 2018

This article has been updated

Abstract

Sensitive detection of off-target effects is important for translating CRISPR–Cas9 nucleases into human therapeutics. In vitro biochemical methods for finding off-targets offer the potential advantages of greater reproducibility and scalability while avoiding limitations associated with strategies that require the culture and manipulation of living cells. Here we describe circularization for in vitro reporting of cleavage effects by sequencing (CIRCLE-seq), a highly sensitive, sequencing-efficient in vitro screening strategy that outperforms existing cell-based or biochemical approaches for identifying CRISPR–Cas9 genome-wide off-target mutations. In contrast to previously described in vitro methods, we show that CIRCLE-seq can be practiced using widely accessible next-generation sequencing technology and does not require reference genome sequences. Importantly, CIRCLE-seq can be used to identify off-target mutations associated with cell-type-specific single-nucleotide polymorphisms, demonstrating the feasibility and importance of generating personalized specificity profiles. CIRCLE-seq provides an accessible, rapid, and comprehensive method for identifying genome-wide off-target mutations of CRISPR–Cas9.

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  • 20 April 2018

    In the version of this Article originally published, the wrong Protocol Exchange DOI and link (10.1038/protex.2017.047) were included in ref. 40. The URL in the reference should have been http://dx.doi.org/10.1038/protex.2017.147. This error has been corrected in the HTML and PDF versions of the paper.

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Sequence Read Archive

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Gene Expression Omnibus

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Acknowledgements

This work was supported by a National Institutes of Health (NIH) Director's Pioneer Award (DP1 GM105378), NIH R35 GM118158, and NIH R01 GM107427 (to J.K.J.); and the Jim and Ann Orr Research Scholar Award (to J.K.J.).

Author information

Author notes

    • Shengdar Q Tsai

    Present address: Department of Hematology, St. Jude Children's Research Hospital, Memphis, Tennessee, USA.

Affiliations

  1. Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, Massachusetts, USA.

    • Shengdar Q Tsai
    • , Nhu T Nguyen
    • , Jose Malagon-Lopez
    • , Ved V Topkar
    • , Martin J Aryee
    •  & J Keith Joung
  2. Center for Cancer Research, Massachusetts General Hospital, Charlestown, Massachusetts, USA.

    • Shengdar Q Tsai
    • , Nhu T Nguyen
    • , Jose Malagon-Lopez
    • , Ved V Topkar
    • , Martin J Aryee
    •  & J Keith Joung
  3. Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts, USA.

    • Shengdar Q Tsai
    • , Nhu T Nguyen
    • , Jose Malagon-Lopez
    • , Ved V Topkar
    • , Martin J Aryee
    •  & J Keith Joung
  4. Department of Pathology, Harvard Medical School, Boston, Massachusetts, USA.

    • Shengdar Q Tsai
    • , Jose Malagon-Lopez
    • , Martin J Aryee
    •  & J Keith Joung
  5. Department of Biostatistics, Harvard T.H. Chan School of Public Health, Boston, Massachusetts, USA.

    • Jose Malagon-Lopez
    •  & Martin J Aryee

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Contributions

S.Q.T. and J.K.J. conceived of and designed experiments. S.Q.T. and N.T.N. performed all experiments. S.Q.T., J.M.-L., V.V.T., and M.J.A. wrote the CIRCLE-seq analysis pipeline and analyzed CIRCLE-seq data. S.Q.T. and J.K.J. wrote the manuscript with input from all authors.

Competing interests

J.K.J. is a consultant for Horizon Discovery. J.K.J. has financial interests in Beacon Genomics, Editas Medicine, Poseida Therapeutics, and Transposagen Biopharmaceuticals. J.K.J.'s interests were reviewed and are managed by Massachusetts General Hospital and Partners HealthCare in accordance with their conflict of interest policies. S.Q.T., M.J.A., and J.K.J. are scientific cofounders of Beacon Genomics.

Corresponding authors

Correspondence to Shengdar Q Tsai or J Keith Joung.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–12, Supplementary Notes 1–2 and Supplementary Table 1.

  2. 2.

    Supplementary Protocol

    Supplementary Protocol: CIRCLE-seq Library Preparation.

Excel files

  1. 1.

    Supplementary Table 2

    List of all CIRCLE-seq detected off-target sites.

  2. 2.

    Supplementary Table 3

    List of CIRCLE-seq read counts and HTGTS scores for off-target sites detected for Cas9 and gRNAs targeted against EMX1 and VEGFA site 1.

  3. 3.

    Supplementary Table 4

    Deep sequencing read counts for targeted tag integration sequencing of off-target cleavage sites of Cas9 and gRNAs targeted against EMX1 and VEGFA site 1.

  4. 4.

    Supplementary Table 5

    Listing of cell-type specific SNPs in protospacer or PAM of off-target cleavage sites detected by CIRCLE-seq.

  5. 5.

    Supplementary Table 6

    Primers used in target tag integration sequencing.

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DOI

https://doi.org/10.1038/nmeth.4278

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