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Photoactivatable CRISPR-Cas9 for optogenetic genome editing

Nature Biotechnology volume 33, pages 755760 (2015) | Download Citation

Abstract

We describe an engineered photoactivatable Cas9 (paCas9) that enables optogenetic control of CRISPR-Cas9 genome editing in human cells. paCas9 consists of split Cas9 fragments and photoinducible dimerization domains named Magnets. In response to blue light irradiation, paCas9 expressed in human embryonic kidney 293T cells induces targeted genome sequence modifications through both nonhomologous end joining and homology-directed repair pathways. Genome editing activity can be switched off simply by extinguishing the light. We also demonstrate activation of paCas9 in spatial patterns determined by the sites of irradiation. Optogenetic control of targeted genome editing should facilitate improved understanding of complex gene networks and could prove useful in biomedical applications.

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References

  1. 1.

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

  2. 2.

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

  3. 3.

    et al. RNA-programmed genome editing in human cells. Elife 2, e00471 (2013).

  4. 4.

    et al. Inducible in vivo genome editing with CRISPR-Cas9. Nat. Biotechnol. 33, 390–394 (2015).

  5. 5.

    et al. An iCRISPR platform for rapid, multiplexable, and inducible genome editing in human pluripotent stem cells. Cell Stem Cell 15, 215–226 (2014).

  6. 6.

    , & A split-Cas9 architecture for inducible genome editing and transcription modulation. Nat. Biotechnol. 33, 139–142 (2015).

  7. 7.

    , , , & Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res. 24, 1012–1019 (2014).

  8. 8.

    et al. Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nat. Biotechnol. 33, 73–80 (2015).

  9. 9.

    et al. Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA. Genome Res. 24, 1020–1027 (2014).

  10. 10.

    & mTOR signaling in growth control and disease. Cell 149, 274–293 (2012).

  11. 11.

    , , , & Light-controlled tools. Angew. Chem. Int. Edn Engl. 51, 8446–8476 (2012).

  12. 12.

    , & Illuminating the chemistry of life: design, synthesis, and applications of “caged” and related photoresponsive compounds. ACS Chem. Biol. 4, 409–427 (2009).

  13. 13.

    , & Manipulating signaling at will: chemically-inducible dimerization (CID) techniques resolve problems in cell biology. Pflugers Arch. 465, 409–417 (2013).

  14. 14.

    et al. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell 156, 935–949 (2014).

  15. 15.

    , , & Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease. Nature 513, 569–573 (2014).

  16. 16.

    et al. Rapid blue-light–mediated induction of protein interactions in living cells. Nat. Methods 7, 973–975 (2010).

  17. 17.

    , , , & Optogenetic protein clustering and signaling activation in mammalian cells. Nat. Methods 10, 249–252 (2013).

  18. 18.

    , , & Engineered pairs of distinct photoswitches for optogenetic control of cellular proteins. Nat. Commun. 6, 6256 (2015).

  19. 19.

    et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol. 31, 827–832 (2013).

  20. 20.

    et al. CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat. Biotechnol. 31, 833–838 (2013).

  21. 21.

    et al. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat. Biotechnol. 31, 822–826 (2013).

  22. 22.

    et al. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 154, 1380–1389 (2013).

  23. 23.

    et al. Surrogate reporters for enrichment of cells with nuclease-induced mutations. Nat. Methods 8, 941–943 (2011).

  24. 24.

    et al. Surrogate reporter-based enrichment of cells containing RNA-guided Cas9 nuclease-induced mutations. Nat. Commun. 5, 3378 (2014).

  25. 25.

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

  26. 26.

    et al. Use of mRNA- and protein-destabilizing elements to develop a highly responsive reporter system. Nucleic Acids Res. 33, e27 (2005).

  27. 27.

    et al. Rapidly reversible manipulation of molecular activity with dual chemical dimerizers. Angew. Chem. Int. Edn Engl. 52, 6450–6454 (2013).

  28. 28.

    et al. Conditional knockouts generated by engineered CRISPR-Cas9 endonuclease reveal the roles of coronin in C. elegans neural development. Dev. Cell 30, 625–636 (2014).

  29. 29.

    , , , & CRISPR-Cas9-based photoactivatable transcription system. Chem. Biol. 22, 169–174 (2015).

  30. 30.

    & A light-inducible CRISPR-Cas9 system for control of endogenous gene activation. Nat. Chem. Biol. 11, 198–200 (2015).

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Acknowledgements

We would like to thank J. Galipon for technical assistance. This work was supported by Platform for Dynamic Approaches to Living System from the Ministry of Education, Culture, Sports, Science and Technology, Japan (MEXT) and by grants from Japan Society for the Promotion of Science (JSPS).

Author information

Affiliations

  1. Graduate School of Arts and Sciences, The University of Tokyo, Komaba, Meguro-ku, Tokyo, Japan.

    • Yuta Nihongaki
    • , Fuun Kawano
    • , Takahiro Nakajima
    •  & Moritoshi Sato

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Contributions

Y.N. and M.S. conceived the project. Y.N., F.K., T.N. and M.S. designed the experiments. Y.N. performed experiments and analyzed data. Y.N. and M.S. wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Moritoshi Sato.

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    Supplementary Text and Figures

    Supplementary Figures 1–8, Supplementary Tables 2 and 3, and Supplementary Notes 1 and 2

Excel files

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    Supplementary Table 1

    Target sequences of sgRNAs and oligonucleotide sequences for constructing sgRNAs.

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DOI

https://doi.org/10.1038/nbt.3245

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