High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells

Journal name:
Nature Biotechnology
Year published:
Published online

Clustered, regularly interspaced, short palindromic repeat (CRISPR) RNA-guided nucleases (RGNs) have rapidly emerged as a facile and efficient platform for genome editing. Here, we use a human cell–based reporter assay to characterize off-target cleavage of CRISPR-associated (Cas)9-based RGNs. We find that single and double mismatches are tolerated to varying degrees depending on their position along the guide RNA (gRNA)-DNA interface. We also readily detected off-target alterations induced by four out of six RGNs targeted to endogenous loci in human cells by examination of partially mismatched sites. The off-target sites we identified harbored up to five mismatches and many were mutagenized with frequencies comparable to (or higher than) those observed at the intended on-target site. Our work demonstrates that RGNs can be highly active even with imperfectly matched RNA-DNA interfaces in human cells, a finding that might confound their use in research and therapeutic applications.


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Author information


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

    • Yanfang Fu,
    • Jennifer A Foden,
    • Cyd Khayter,
    • Morgan L Maeder,
    • Deepak Reyon,
    • J Keith Joung &
    • Jeffry D Sander
  2. Center for Cancer Research, Massachusetts General Hospital, Charlestown, Massachusetts, USA.

    • Yanfang Fu,
    • Jennifer A Foden,
    • Cyd Khayter,
    • Morgan L Maeder,
    • Deepak Reyon,
    • J Keith Joung &
    • Jeffry D Sander
  3. Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts, USA.

    • Yanfang Fu,
    • Jennifer A Foden,
    • Cyd Khayter,
    • Morgan L Maeder,
    • Deepak Reyon,
    • J Keith Joung &
    • Jeffry D Sander
  4. Department of Pathology, Harvard Medical School, Boston, Massachusetts, USA.

    • Yanfang Fu,
    • Deepak Reyon,
    • J Keith Joung &
    • Jeffry D Sander
  5. Program in Biological and Biomedical Sciences, Harvard Medical School, Boston, Massachusetts, USA.

    • Morgan L Maeder &
    • J Keith Joung


Y.F., J.D.S. and J.K.J. designed experiments; Y.F., J.A.F., C.K., M.L.M., D.R. and J.D.S. performed experiments; Y.F., M.L.M., D.R., J.D.S. and J.K.J. wrote the manuscript.

Competing financial interests

J.K.J. has a financial interest in 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.

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Supplementary information

PDF files

  1. Supplementary Text and Figures (61 MB)

    Supplementary Figures 1–14, Supplementary Note and Supplementary Methods

Excel files

  1. Supplementary Table 1 (37 KB)

    Sequences of oligonucleotides used to generate expression plasmids encoding sgRNAs/variant sgRNAs targeted to sites in the EGFP reporter gene and sgRNAs targeted to six endogenous human gene targets

  2. Supplementary Table 2 (53 KB)

    Sequences and characteristics of genomic on- and off-target sites for six RGENs targeted to endogenous human genes and primers and PCR conditions used to amplify these sites

Additional data