Directly modulating the activity of genome-editing proteins has the potential to increase their specificity by reducing activity following target locus modification. We developed Cas9 nucleases that are activated by the presence of a cell-permeable small molecule by inserting an evolved 4-hydroxytamoxifen–responsive intein at specific positions in Cas9. In human cells, conditionally active Cas9s modify target genomic sites with up to 25-fold higher specificity than wild-type Cas9.
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This work was supported by National Institutes of Health R01 GM095501, Defense Advanced Research Projects Agency HR0011-11-2-0003 and N66001-12-C-4207 and the Howard Hughes Medical Institute. K.M.D. acknowledges the Natural Sciences and Engineering Research Council of Canada (NSERC) for a Postgraduate Scholarship–Doctoral (PGS D) award. J.A.Z. is a Ruth L. Kirchstein National Research Service Awards Postdoctoral Fellow (F32 GM 106601-2). We are grateful to J. Doudna, S. Sternberg, D. Taylor, M. Jinek and F. Jiang for providing the structural coordinates of Cas9 and to J. Guilinger, Y. Kim, M. Li and A. Badran for helpful discussions.
The co-authors have filed a provisional patent application related to this work. D.R.L is a consultant for Editas Medicine, a company that applies genome-editing technologies.
Supplementary Results, Supplementary Figures 1–8, Supplementary Tables 1–7 and Supplementary Notes. (PDF 1802 kb)
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Davis, K., Pattanayak, V., Thompson, D. et al. Small molecule–triggered Cas9 protein with improved genome-editing specificity. Nat Chem Biol 11, 316–318 (2015). https://doi.org/10.1038/nchembio.1793
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