Article | Published:

DNA-binding-domain fusions enhance the targeting range and precision of Cas9

Nature Methods volume 12, pages 11501156 (2015) | Download Citation

Abstract

The CRISPR-Cas9 system is commonly used in biomedical research; however, the precision of Cas9 is suboptimal for applications that involve editing a large population of cells (for example, gene therapy). Variations on the standard Cas9 system have yielded improvements in the precision of targeted DNA cleavage, but they often restrict the range of targetable sequences. It remains unclear whether these variants can limit lesions to a single site in the human genome over a large cohort of treated cells. Here we show that by fusing a programmable DNA-binding domain (pDBD) to Cas9 and attenuating Cas9’s inherent DNA-binding affinity, we were able to produce a Cas9-pDBD chimera with dramatically improved precision and an increased targeting range. Because the specificity and affinity of this framework can be easily tuned, Cas9-pDBDs provide a flexible system that can be tailored to achieve extremely precise genome editing at nearly any genomic locus.

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Acknowledgements

We thank M. Porteus (Stanford Medicine, Stanford, California, USA) for GFP reporter vector M427, N. Rhind for the use of his FACS machine, E. Kittler and the UMass Medical School Deep Sequencing Core for their assistance with the Illumina sequencing, and E. Sontheimer for insightful discussions. All new reagents described in this work have been deposited with the nonprofit plasmid-distribution service Addgene. This work was supported by the US National Institutes of Health (grant R01AI117839 to S.A.W. and J. Luban, grant U01HG007910 to M.G. and J. Luban, and grant R01HL093766 to S.A.W. and N. Lawson).

Author information

Affiliations

  1. Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, Massachusetts, USA.

    • Mehmet Fatih Bolukbasi
    • , Ankit Gupta
    • , Sarah Oikemus
    • , Michael H Brodsky
    • , Lihua Julie Zhu
    •  & Scot A Wolfe
  2. Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts, USA.

    • Mehmet Fatih Bolukbasi
    •  & Scot A Wolfe
  3. Program in Bioinformatics and Integrative Biology, University of Massachusetts Medical School, Worcester, Massachusetts, USA.

    • Alan G Derr
    • , Manuel Garber
    •  & Lihua Julie Zhu
  4. Department of Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts, USA.

    • Manuel Garber
    •  & Lihua Julie Zhu

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Contributions

M.F.B. and A.G. performed all cell-based experiments. A.G.D., M.G. and L.J.Z. performed the bioinformatic analysis. S.O. and M.H.B. optimized the GFP reporter assay. M.F.B., A.G., L.J.Z. and S.A.W. directed the research and interpreted experiments. M.F.B., A.G. and S.A.W. wrote the manuscript with input from all the other authors.

Competing interests

The authors have filed patent applications related to genome engineering technologies. S.A.W. is a consultant for Editas Medicine.

Corresponding author

Correspondence to Scot A Wolfe.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–14, Supplementary Tables 1–2, Supplementary Note and Supplementary Discussion

Excel files

  1. 1.

    Supplementary Table 3

    Summary of lesion rates determined through targeted PCR-based deep-sequencing of potential off-target sites

  2. 2.

    Supplementary Table 4

    List of primers and on/off target sequences used in this study

  3. 3.

    Supplementary Table 5

    List of indexes used to identify genomic regions for targeted PCR deep-sequencing analysis

  4. 4.

    Supplementary Table 6

    Summary of peaks detected from GUIDE-seq off-target analysis

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DOI

https://doi.org/10.1038/nmeth.3624