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.
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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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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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Supplementary Information
Supplementary Results, Supplementary Figures 1–13, Supplementary Tables 1–4, Supplementary Sequences, Supplementary Database, Supplementary Methods, Supplementary References.
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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DOI: https://doi.org/10.1038/s41477-017-0046-0
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