Letter | Published:

CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering

Nature Biotechnology volume 31, pages 833838 (2013) | Download Citation

Abstract

Prokaryotic type II CRISPR-Cas systems can be adapted to enable targeted genome modifications across a range of eukaryotes1,2,3,4,5,6,7. Here we engineer this system to enable RNA-guided genome regulation in human cells by tethering transcriptional activation domains either directly to a nuclease-null Cas9 protein or to an aptamer-modified single guide RNA (sgRNA). Using this functionality we developed a transcriptional activation–based assay to determine the landscape of off-target binding of sgRNA:Cas9 complexes and compared it with the off-target activity of transcription activator–like (TALs) effectors8,9. Our results reveal that specificity profiles are sgRNA dependent, and that sgRNA:Cas9 complexes and 18-mer TAL effectors can potentially tolerate 1–3 and 1–2 target mismatches, respectively. By engineering a requirement for cooperativity through offset nicking for genome editing or through multiple synergistic sgRNAs for robust transcriptional activation, we suggest methods to mitigate off-target phenomena. Our results expand the versatility of the sgRNA:Cas9 tool and highlight the critical need to engineer improved specificity.

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Acknowledgements

P.M. thanks R. Kalhor for insightful discussions. This work was supported by US National Institutes of Health grant P50 HG005550 and Department of Energy grant DE-FG02-02ER63445.

Author information

Author notes

    • Prashant Mali
    •  & John Aach

    These authors contributed equally to this work.

Affiliations

  1. Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.

    • Prashant Mali
    • , John Aach
    • , P Benjamin Stranges
    • , Mark Moosburner
    •  & George M Church
  2. Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, Massachusetts, USA.

    • Kevin M Esvelt
    • , Sriram Kosuri
    •  & George M Church
  3. Biological and Biomedical Sciences Program, Harvard Medical School, Boston, Massachusetts, USA.

    • Luhan Yang

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Contributions

P.M., J.A. and G.M.C. conceived the study. P.M. designed and performed experiments. J.A. designed and performed bioinformatic analyses. M.M. performed experiments. P.B.S., K.M.E., S.K. and L.Y. developed reagents and performed analyses. P.M., J.A. and G.M.C. wrote the manuscript with support from all authors.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to George M Church.

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    Supplementary Figures 1–17, Supplementary Tables 1–3 and Supplementary Notes 1–3

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DOI

https://doi.org/10.1038/nbt.2675

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