LETTERS https://doi.org/10.1038/s41477-017-0046-0 A potent Cas9-derived gene activator for plant and mammalian cells Zhenxiang Li1, Dandan Zhang1, Xiangyu Xiong1, Bingyu Yan1, Wei Xie1,2, Jen Sheen3 and Jian-Feng Li 1,2,4* Overexpression of complementary DNA represents the most presumably by promoting the transcription initiation of the sgRNA commonly used gain-of-function approach for interrogat- by the U6 promoter. Therefore, we routinely add a G to the 5′ end ing gene functions and for manipulating biological traits. of sgRNAs when the target sequences start with a non-G nucleotide. However, this approach is challenging and inefficient for Although multiple sgRNAs tiling the proximal promoter of the multigene expression due to increased labour for cloning, target gene can synergistically boost the dCas9–VP64-mediated limited vector capacity, requirement of multiple promoters gene activation7–9,12–15, this strategy reduces the scalability of the sys- and terminators, and variable transgene expression levels. tem2 and may increase the risk of dCas9-mediated transcriptional Synthetic transcriptional activators provide a promising alter- perturbation at off-target non-promoter loci15–17. Therefore, we native strategy for gene activation by tethering an autono- sought to devise and screen for an improved dCas9–TAD (Fig. 1a) mous transcription activation domain (TAD) to an intended that would allow potent transcriptional activation of target genes gene promoter at the endogenous genomic locus through a using only a single sgRNA, thus maximizing the system’s scalabil- programmable DNA-binding module. Among the known cus- ity. As the first step, we modified dCas9–VP64 by adding additional tom DNA-binding modules, the nuclease-dead Streptococcus VP64 moieties (Supplementary Sequences). By using a WRKY30 pyogenes Cas9 (dCas9) protein, which recognizes a spe- promoter–LUC reporter and a pre-screened optimal sgRNA cific DNA target through base pairing between a synthetic (WRKY30-2, Supplementary Fig. 2a), we observed that dCas9– guide RNA and DNA, outperforms zinc-finger proteins and VP128 outperformed dCas9–VP64 by increasing the LUC activa- transcription activator-like effectors, both of which target tion from twofold to more than fivefold relative to the basal level through protein–DNA interactions1. Recently, three potent (Fig. 1b). In contrast, dCas9–VP192 and dCas9–VP256 exhibited dCas9-based transcriptional activation systems, namely a sharp decrease of protein production and severe protein degrada- VPR, SAM and SunTag, have been developed for animal tion, leading to an overall reduced LUC activation (Fig. 1b). The cells2–6. However, an efficient dCas9-based transcriptional fact that VP192 and VP256 correspond to 12 and 16 repeats of the activation platform is still lacking for plant cells7–9. Here, VP16 motif11, respectively, suggests that their expression and stabil- we developed a new potent dCas9–TAD, named dCas9–TV, ity issues may be incurred by highly repetitive sequences. through plant cell-based screens. dCas9–TV confers far stron- As a second step to enhance the activity of dCas9–VP128, we ger transcriptional activation of single or multiple target incorporated other sequence-unrelated portable TADs, which genes than the routinely used dCas9–VP64 activator in both included plant-specific EDLL18 and ERF2m19 (modified ERF2) plant and mammalian cells. motifs as well as a TAD from Xanthamonas TALEs20. To minimize Among synthetic gene activators, dCas9–TADs potentially offer potential interference between different TADs, flexible GGSGG unparalleled simplicity and multiplexability compared with zinc- linkers were used as spacers between TADs (Supplementary finger protein–TADs and transcription activator-like effector Sequences). We observed that the combination of VP128 with up to (TALE)–TADs because synthetic guide RNAs (sgRNAs) can be four copies of tandem ERF2m–EDLL motifs (hereafter referred to as easily modified to achieve new targeting specificities, and dCas9 EE) activated LUC expression by 12.6-fold relative to the basal level guided by multiple sgRNAs can simultaneously bind to several dif- (Fig. 1c), and the combination of VP128 with up to six copies of the ferent target loci10. However, a dCas9 fusion with VP64, a frequently TALE TAD motif (hereafter referred to as TAL) exhibited a maximal used TAD11, only weakly activates target genes using a single sgRNA activation of LUC expression by more than 55-fold (Fig. 1c). Of note, in plant and mammalian cells7–9,12–15. Using Arabidopsis proto- further insertion of TAL motifs triggered severe protein degrada- plast-based promoter–luciferase (LUC) assays, we confirmed that tion, presumably due to the high sequence repetition, resulting in an dCas9–VP64 with a single sgRNA only weakly (maximally 2.4-fold) overall decreased LUC induction (Fig. 1c). Therefore, the cell-based or ineffectively activated target genes (Supplementary Results and screens identified dCas9–6TAL–VP128 as a potentially strong tran- Supplementary Figs. 1 and 2). Interestingly, when the target sequence scription activator, which was renamed dCas9–TV for simplicity. lacks a 5′ G, an extra G appended to the 5′ end of the sgRNA was In addition, when we used an Arabidopsis U6-26 rather than U6-1 found to enhance the promoter activation (Supplementary Fig. 1), promoter to express the sgRNA-WRKY30-2, we detected a further 1Key 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. 2Guangzhou Key Laboratory of Healthy Aging Research, School of Life Sciences, Sun Yat-sen University, Guangzhou, China. 3Department of Molecular Biology and Centre for Computational and Integrative Biology, Massachusetts General Hospital, and Department of Genetics, Harvard Medical School, Boston, MA, USA. 4Collaborative Innovation Center of Genetics and Development, School of Life Sciences, Sun Yat-sen University, Guangzhou, China. Zhenxiang Li and Dandan Zhang contributed equally to this work. *e-mail: [email protected] 930 NATURE PLANTS | VOL 3 | DECEMBER 2017 | 930–936 | www.nature.com/natureplants © 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. © 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. NATURE PLANTS LETTERS a b c 70 AtWRKY30–LUC ** * 55.6 52.1 dCas9–VP64 AtWRKY30–LUC 60 7 dCas9 * 6 5.1 50 5 40 * dCas9–4EE–VP128 3.3* 27.1 4 30 dCas9 3 * NS * 2.0 20 * 12.6 2 1.4 * 9.8 9.6* 1.0 6.9** 8.0 dCas9–6TAL–VP128 1 10 Promoter activity (RLU) 1.0 0 0 dCas9 Promoter activity (RLU) Ctrl Ctrl VP64 VP128 VP192 VP256 VP128 dCas9 EE–VP128 2 FLAG 2EE–VP1284EE–VP128 × NLS VP64 ERF2m EDLL TAL * 2TAL–VP1284TAL–VP1286TAL–VP1288TAL–VP128 dCas9 * d e 60 AtRLP23 44.2 f 40 AtRLP23 32.3 180 31.5 AtWRKY30 30.4 31.4 160 138.8 50 35 140 30 120 40 100 25 80 30 20 60 47.5 15 40 20 20 10 Relative gene expression 1.0 2.1 1.1 Relative gene expression 10 Relative gene expression 0 5 1.0 0.7 1.0 0.5 0.9 0.5 WT no. 13 no. 15 no. 19 no. 29 0 0 Ctrl VP64 TV WT no. 1 no. 4 no. 5 no. 1 no. 9 no. 10 no. 14 dCas9–VP64 dCas9–TV dCas9 dCas9–VP64 dCas9–TV gh4,000 3,500 WT WT no. 1 no. 9 no. 10 no. 14 no. 1 3,000 no. 9 10 nM nlp20 –++ –+–+–+– 2,500 no. 10 MPK6 MPK3 2,000 no. 14 MPK4 1,500 ROS burst RuBisCo 1,000 500 0 0 51015202530 35 40 45 50 i 4 Activation Time (min) R = 0.993 j 250 3 AtWRKY30 AtRLP23 191.7 (FPKM+1)) 200 AtCDG1 10 2 150 1 AtRLP23 100 80.3 37.3 dCas9–TV (log Repression 50 0 Relative gene expression 1.0 1.0 1.0 0.9 1.3 3.2 01234 0 Ctrl (log10(FPKM+1)) Ctrl dCas9–VP64 dCas9–TV Fig. 1 | dCas9–TV-mediated gene activation in Arabidopsis a, Diagram of three representative dCas9 gene activators tested in this study. FLAG, FLAG tag; NLS, nuclear localization signal. b, dCas9–VP128 exhibits the highest activity among dCas9–(VP64)n (n = 1–4) activators in Arabidopsis protoplasts. dCas9–VP256 is marked by an asterisk in the blot. c, dCas9–6TAL–VP128 (dCas9–TV) exhibits the highest activity among all screened dCas9 activators in Arabidopsis protoplasts. dCas9–8TAL–VP128 is marked by an asterisk in the blot. d, Representative transgenic Arabidopsis T1 plants co-expressing dCas9–TV and sgRNA-WRKY30 show strong induction of endogenous WRKY30. e,f, Arabidopsis protoplasts (e) or representative transgenic T2 plants (f) co-expressing dCas9–TV and sgRNA-RLP23 show robust induction of endogenous RLP23. g,h, Arabidopsis transgenic T2 plants with RLP23 induction accordingly exhibit enhanced MAPK activation (g) and reactive oxygen species (ROS) production (h) in response to the pathogen elicitor nlp20. RuBisCo in g indicates equal protein loading. i, Evaluation of dCas9–TV specificity by RNA-seq. Arabidopsis protoplasts expressing or not expressing dCas9–TV and sgRNA-RLP23 exhibit very similar transcriptome profiles. R indicates Pearson’s correlation coefficient. AtRLP23 is marked by a red dot close to the y axis as a highly activated gene. j, Co-expression of dCas9–TV and three sgRNAs leads to simultaneous induction of WRKY30, RLP23 and CDG1 in Arabidopsis protoplasts. Empty vectors were used in control (Ctrl) samples to replace constructs expressing dCas9 activator and sgRNAs. Gene activation was determined by promoter–LUC assays (b,c) or RT-qPCR (d–f,j), and data are shown as mean (indicated with a number above the bar) ± s.d. (n = 3). *P < 0.05, **P < 0.01 (Student’s t-test). NS, not significant; WT, wild type; RLU, relative luciferase unit; At, Arabidopsis thaliana.