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

Nature Biotechnology volume 31, pages 833–838 (2013)Cite this article

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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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Figure 1: RNA-guided transcriptional activation.
Figure 2: Evaluating the landscape of targeting by sgRNA:Cas9 complexes and TAL effectors.
Figure 3: Offset nicking.

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References

  1. Cho, S.W., Kim, S., Kim, J.M. & Kim, J.S. Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat. Biotechnol. 31, 230–232 (2013).

    Article  CAS  Google Scholar 

  2. Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).

    Article  CAS  Google Scholar 

  3. DiCarlo, J.E. et al. Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acids Res. 41, 4336–4343 (2013).

    Article  CAS  Google Scholar 

  4. Hwang, W.Y. et al. Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat. Biotechnol. 31, 227–229 (2013).

    Article  CAS  Google Scholar 

  5. Jinek, M. et al. RNA-programmed genome editing in human cells. eLife 2, e00471 (2013).

    Article  Google Scholar 

  6. Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013).

    Article  CAS  Google Scholar 

  7. Qi, L.S. et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152, 1173–1183 (2013).

    Article  CAS  Google Scholar 

  8. Boch, J. et al. Breaking the code of DNA binding specificity of TAL-type III effectors. Science 326, 1509–1512 (2009).

    Article  CAS  Google Scholar 

  9. Moscou, M.J. & Bogdanove, A.J. A simple cipher governs DNA recognition by TAL effectors. Science 326, 1501 (2009).

    Article  CAS  Google Scholar 

  10. Deltcheva, E. et al. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature 471, 602–607 (2011).

    Article  CAS  Google Scholar 

  11. Gasiunas, G., Barrangou, R., Horvath, P. & Siksnys, V. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc. Natl. Acad. Sci. USA 109, E2579–E2586 (2012).

    Article  CAS  Google Scholar 

  12. Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).

    Article  CAS  Google Scholar 

  13. Sapranauskas, R. et al. The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli. Nucleic Acids Res. 39, 9275–9282 (2011).

    Article  CAS  Google Scholar 

  14. Bhaya, D., Davison, M. & Barrangou, R. CRISPR-Cas systems in bacteria and archaea: versatile small RNAs for adaptive defense and regulation. Annu. Rev. Genet. 45, 273–297 (2011).

    Article  CAS  Google Scholar 

  15. Zhang, F. et al. Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription. Nat. Biotechnol. 29, 149–153 (2011).

    Article  Google Scholar 

  16. Fusco, D. et al. Single mRNA molecules demonstrate probabilistic movement in living mammalian cells. Curr. Biol. 13, 161–167 (2003).

    Article  CAS  Google Scholar 

  17. Takahashi, K. et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861–872 (2007).

    Article  CAS  Google Scholar 

  18. Maeder, M.L. et al. Robust, synergistic regulation of human gene expression using TALE activators. Nat. Methods 10, 243–245 (2013).

    Article  CAS  Google Scholar 

  19. Perez-Pinera, P. et al. Synergistic and tunable human gene activation by combinations of synthetic transcription factors. Nat. Methods 10, 239–242 (2013).

    Article  CAS  Google Scholar 

  20. Jiang, W., Bikard, D., Cox, D., Zhang, F. & Marraffini, L.A. RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat. Biotechnol. 31, 233–239 (2013).

    Article  CAS  Google Scholar 

  21. Patwardhan, R.P. et al. Massively parallel functional dissection of mammalian enhancers in vivo. Nat. Biotechnol. 30, 265–270 (2012).

    Article  CAS  Google Scholar 

  22. Sanjana, N.E. et al. A transcription activator-like effector toolbox for genome engineering. Nat. Protoc. 7, 171–192 (2012).

    Article  CAS  Google Scholar 

  23. Meckler, J.F. et al. Quantitative analysis of TALE-DNA interactions suggests polarity effects. Nucleic Acids Res. 41, 4118–4128 (2013).

    Article  CAS  Google Scholar 

  24. Reyon, D. et al. FLASH assembly of TAL effectors for high-throughput genome editing. Nat. Biotechnol. 30, 460–465 (2012).

    Article  CAS  Google Scholar 

  25. Streubel, J., Blucher, C., Landgraf, A. & Boch, J. TAL effector RVD specificities and efficiencies. Nat. Biotechnol. 30, 593–595 (2012).

    Article  CAS  Google Scholar 

  26. Porteus, M.H. & Carroll, D. Gene targeting using zinc finger nucleases. Nat. Biotechnol. 23, 967–973 (2005).

    Article  CAS  Google Scholar 

  27. Pattanayak, V., Ramirez, C.L., Joung, J.K. & Liu, D.R. Revealing off-target cleavage specificities of zinc-finger nucleases by in vitro selection. Nat. Methods 8, 765–770 (2011).

    Article  CAS  Google Scholar 

  28. Gabriel, R. et al. An unbiased genome-wide analysis of zinc-finger nuclease specificity. Nat. Biotechnol. 29, 816–823 (2011).

    Article  CAS  Google Scholar 

  29. Certo, M.T. et al. Tracking genome engineering outcome at individual DNA breakpoints. Nat. Methods 8, 671–676 (2011).

    Article  CAS  Google Scholar 

  30. Symington, L.S. & Gautier, J. Double-strand break end resection and repair pathway choice. Annu. Rev. Genet. 45, 247–271 (2011).

    Article  CAS  Google Scholar 

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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.

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Author notes

  1. Prashant Mali and John Aach: These authors contributed equally to this work.

Authors and 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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  1. Prashant Mali
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  4. Kevin M Esvelt
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  8. George M Church
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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.

Corresponding author

Correspondence to George M Church.

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The authors declare no competing financial interests.

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Supplementary Figures 1–17, Supplementary Tables 1–3 and Supplementary Notes 1–3 (PDF 28595 kb)

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Mali, P., Aach, J., Stranges, P. et al. CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat Biotechnol 31, 833–838 (2013). https://doi.org/10.1038/nbt.2675

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