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A potent Cas9-derived gene activator for plant and mammalian cells

Nature Plantsvolume 3pages930936 (2017) | Download Citation


Overexpression of complementary DNA represents the most commonly used gain-of-function approach for interrogating gene functions and for manipulating biological traits. However, this approach is challenging and inefficient for multigene expression due to increased labour for cloning, limited vector capacity, requirement of multiple promoters and terminators, and variable transgene expression levels. Synthetic transcriptional activators provide a promising alternative strategy for gene activation by tethering an autonomous transcription activation domain (TAD) to an intended gene promoter at the endogenous genomic locus through a programmable DNA-binding module. Among the known custom DNA-binding modules, the nuclease-dead Streptococcus pyogenes Cas9 (dCas9) protein, which recognizes a specific DNA target through base pairing between a synthetic guide RNA and DNA, outperforms zinc-finger proteins and transcription activator-like effectors, both of which target through protein–DNA interactions1. Recently, three potent dCas9-based transcriptional activation systems, namely VPR, SAM and SunTag, have been developed for animal cells2,3,4,5,6. However, an efficient dCas9-based transcriptional activation platform is still lacking for plant cells7,8,9. Here, we developed a new potent dCas9–TAD, named dCas9–TV, through plant cell-based screens. dCas9–TV confers far stronger transcriptional activation of single or multiple target genes than the routinely used dCas9–VP64 activator in both plant and mammalian cells.

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  1. 1.

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

  2. 2.

    Konermann, S. et al. Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Nature 517, 583–588 (2015).

  3. 3.

    Tanenbaum, M. E., Gilbert, L. A., Qi, L. S., Weissman, J. S. & Vale, R. D. A protein-tagging system for signal amplification in gene expression and fluorescence imaging. Cell 159, 635–646 (2014).

  4. 4.

    Gilbert, L. A. et al. Genome-scale CRISPR-mediated control of gene repression and activation. Cell 159, 647–661 (2014).

  5. 5.

    Chavez, A. et al. Highly efficient Cas9-mediated transcriptional programming. Nat. Methods 12, 2–6 (2015).

  6. 6.

    Chavez, A. et al. Comparison of Cas9 activators in multiple species. Nat. Methods 13, 563–567 (2016).

  7. 7.

    Piatek, A. et al. RNA-guided transcriptional regulation in planta via synthetic dCas9-based transcription factors. Plant Biotechnol. J. 13, 578–589 (2015).

  8. 8.

    Lowder, L. G. et al. A CRISPR/Cas9 toolbox for multiplexed plant genome editing and transcriptional regulation. Plant Physiol. 169, 971–985 (2015).

  9. 9.

    Vazquez-Vilar, M. et al. A modular toolbox for gRNA-Cas9 genome engineering in plants based on the GoldenBraid standard. Plant Methods 12, 10 (2016).

  10. 10.

    Didovyk, A., Borek, B., Tsimring, L. & Hasty, J. Transcriptional regulation with CRISPR-Cas9: principles, advances, and applications. Curr. Opin. Biotechnol. 40, 177–184 (2016).

  11. 11.

    Beerli, R. R., Segal, D. J., Dreier, B. & Barbas, C. F. III Toward controlling gene expression at will: Specific regulation of the erbB-2/HER-2 promoter by using polydactyl zinc finger proteins constructed from modular building blocks. Proc. Natl Acad. Sci. USA 95, 14628–14633 (1998).

  12. 12.

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

  13. 13.

    Perez-Pinera, P. et al. RNA-guided gene activation by CRISPR-Cas9-based transcription factors. Nat. Methods 10, 973–976 (2013).

  14. 14.

    Maeder, M. L. et al. CRISPR RNA-guided activation of endogenous human genes. Nat. Methods 10, 977–979 (2013).

  15. 15.

    Cheng, A. W. et al. Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system. Cell Res. 23, 1163–1171 (2013).

  16. 16.

    Farzadfard, F., Perli, S. D. & Lu, T. K. Tunable and multifunctional eukaryotic transcription factors based on CRISPR/Cas. ACS Synth. Biol. 2, 604–613 (2013).

  17. 17.

    Braun, C. J. et al. Versatile in vivo regulation of tumor phenotypes by dCas9-mediated transcriptional perturbation. Proc. Natl Acad. Sci. USA 113, 3892–3900 (2016).

  18. 18.

    Tiwari, S. B. et al. The EDLL motif: a potent plant transcriptional activation domain from AP2/ERF transcription factors. Plant J. 70, 855–865 (2012).

  19. 19.

    Li, J. et al. Activation domains for controlling plant gene expression using designed transcription factors. Plant Biotechnol. J. 11, 671–680 (2013).

  20. 20.

    Zhu, W., Yang, B., Wills, N., Johnson, L. B. & White, F. F. The C terminus of AvrXa10 can be replaced by the transcriptional activation domain of VP16 from the herpes simplex virus. Plant Cell 11, 1665–1674 (1999).

  21. 21.

    Tang, X. et al. A CRISPR-Cpf1 system for efficient genome editing and transcriptional repression in plants. Nat. Plants 3, 17018 (2017).

  22. 22.

    Zipfel, C. & Oldroyd, G. E. D. Plant signalling in symbiosis and immunity. Nature 543, 328–336 (2017).

  23. 23.

    Hu, J. et al. Direct activation of human and mouse Oct4 genes using engineered TALE and Cas9 transcription factors. Nucleic Acids Res. 42, 4375–4390 (2014).

  24. 24.

    Li, J. F., Zhang, D. & Sheen, J. Epitope-tagged protein-based artificial miRNA screens for optimized gene silencing in plants. Nat. Protoc. 9, 939–949 (2015).

  25. 25.

    Puchta, H. Using CRISPR/Cas in three dimensions: towards synthetic plant genomes, transcriptomes and epigenomes. Plant J. 87, 5–15 (2016).

  26. 26.

    Schellenberger, V. et al. A recombinant polypeptide extends the in vivo half-life of peptides and proteins in a tunable manner. Nat. Biotechnol. 27, 1186–1190 (2009).

  27. 27.

    Ryu, J. et al. Protein-stabilizing and cell-penetrating properties of α-helix domain of 30Kc19 protein. Biotechnol. J. 11, 1443–1451 (2016).

  28. 28.

    Li, J. F. et al. Multiplex and homologous recombination–mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9. Nat. Biotechnol. 31, 688–691 (2013).

  29. 29.

    Li, J. F. et al. Comprehensive protein-based artificial microRNA screens for effective gene silencing in plants. Plant Cell 25, 1507–1522 (2013).

  30. 30.

    Zetsche, B. et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell 163, 759–771 (2015).

  31. 31.

    Zetsche, B. et al. Multiplex gene editing by CRISPR–Cpf1 using a single crRNA array. Nat. Biotechnol. 35, 31–34 (2016).

  32. 32.

    Zhang, Y. et al. A highly efficient rice green tissue protoplast system for transient gene expression and studying light/chloroplast-related processes. Plant Methods 7, 30 (2011).

  33. 33.

    Clough, S. J. & Bent, A. F. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16, 735–743 (1998).

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We thank F. Ausubel and Z. Cheng for critical reading of this manuscript. This work was supported by the National Natural Science Foundation of China grant 31522006 and start-up funds from China’s Thousand Young Talents Program to J.-F.L. and the NIH grant R01GM70567 to J.S. This work was partially supported by the Guangzhou Science and Technology Project grant 201605030012.

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Author notes

  1. Zhenxiang Li and Dandan Zhang contributed equally to this work.


  1. Key Laboratory of Gene Engineering of Ministry of Education, State Key Laboratory of Biocontrol, Guangdong Provincial Key Laboratory of Plant Resources, School of Life Sciences, Sun Yat-sen University, Guangzhou, China

    • Zhenxiang Li
    • , Dandan Zhang
    • , Xiangyu Xiong
    • , Bingyu Yan
    • , Wei Xie
    •  & Jian-Feng Li
  2. Guangzhou Key Laboratory of Healthy Aging Research, School of Life Sciences, Sun Yat-sen University, Guangzhou, China

    • Wei Xie
    •  & Jian-Feng Li
  3. Department of Molecular Biology and Centre for Computational and Integrative Biology, Massachusetts General Hospital, and Department of Genetics, Harvard Medical School, Boston, MA, USA

    • Jen Sheen
  4. Collaborative Innovation Center of Genetics and Development, School of Life Sciences, Sun Yat-sen University, Guangzhou, China

    • Jian-Feng Li


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J.-F.L. and J.S. conceived the study. J.-F.L. designed the experiments and supervised the study. D.Z. conducted the protoplast-based screens of dCas9 activators. Z.L. conducted other dCas9–TV experiments in Arabidopsis protoplasts and transgenic plants. X.X. and Z.L. conducted the dCas9–TV experiments in rice protoplasts. B.Y. conducted the dCas9–TV experiments in human HEK 293T cells. Z.L., X.X. and W.X. performed the RNP-mediated gene activation. J.-F.L. wrote the manuscript with input from J.S. and all other authors.

Competing interests

The authors have filed a patent application based on some results reported in this paper.

Corresponding author

Correspondence to Jian-Feng Li.

Electronic supplementary material

  1. Supplementary Information

    Supplementary Results, Supplementary Figures 1–13, Supplementary Tables 1–4, Supplementary Sequences, Supplementary Database, Supplementary Methods, Supplementary References.

  2. Life Sciences Reporting Summary

  3. Supplementary Dataset 1

    RNA-seq data of Arabidopsis protoplasts expressing or not expressing dCas9–TV and sgRNA-RLP23.

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