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

Nature Plants volume 3, pages 930–936 (2017)Cite this article

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Abstract

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.

Among synthetic gene activators, dCas9–TADs potentially offer unparalleled simplicity and multiplexability compared with zinc-finger protein–TADs and transcription activator-like effector (TALE)–TADs because synthetic guide RNAs (sgRNAs) can be easily modified to achieve new targeting specificities, and dCas9 guided by multiple sgRNAs can simultaneously bind to several different target loci10. However, a dCas9 fusion with VP64, a frequently used TAD11, only weakly activates target genes using a single sgRNA in plant and mammalian cells7,8,9,12,13,14,15. Using Arabidopsis protoplast-based promoter–luciferase (LUC) assays, we confirmed that dCas9–VP64 with a single sgRNA only weakly (maximally 2.4-fold) or ineffectively activated target genes (Supplementary Results and Supplementary Figs. 1 and 2). Interestingly, when the target sequence lacks a 5′ G, an extra G appended to the 5′ end of the sgRNA was found to enhance the promoter activation (Supplementary Fig. 1), presumably by promoting the transcription initiation of the sgRNA by the U6 promoter. Therefore, we routinely add a G to the 5′ end of sgRNAs when the target sequences start with a non-G nucleotide.

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Fig. 1: dCas9–TV-mediated gene activation in Arabidopsis
Fig. 2: Modified strategies of dCas9–TV-mediated gene activation in Arabidopsis and other cells.

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Acknowledgements

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.

Authors and Affiliations

  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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Contributions

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.

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Correspondence to Jian-Feng Li.

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

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Supplementary Information

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

Life Sciences Reporting Summary

Supplementary Dataset 1

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

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Li, Z., Zhang, D., Xiong, X. et al. A potent Cas9-derived gene activator for plant and mammalian cells. Nature Plants 3, 930–936 (2017). https://doi.org/10.1038/s41477-017-0046-0

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